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Detection of Pathogenic Aeromonas hydrophila from Rainbow Trout (Oncorhynchus mykiss) Farms in Turkey.

Byline: Ifakat Tulay Cagatay and Evrim Beyhan Sen


The aim of this study was identifying and detecting virulent Aeromonas hydrophila, which caused bacterial hemorrhagic septicemia in rainbow trout (Oncorhynchus mykiss) farms in Mugla-Fethiye region by using PCR amplification of virulence- related genes. For this reason, clinically observed diseased fish samples were collected between 2009 and 2011. Three target genes, cytolytic enterotoxin (AHCYTOEN), hemolysin (Hly) and bacterial outer membrane protein (OmpTS) genes have been chosen for detecting virulent bacteria from those samples. Detected A. hydrophila from infected fish samples was demonstrated with positive amplifications of three genes at the same time. In addition, these positive results were separately confirmed by the amplification results of references strains. As a result, the infectious agent of this disease was identified rapidly by using the direct detection of bacteria through specific virulence determinants as genetic markers, before it spreads out. Copyright 2014 Friends Science Publishers

Keywords: Aeromonas hydrophila; AHCYTOEN; Hly; OmpTSIntroduction

Aeromonas hydrophila is a gram negative, an opportunistic and zoonatically important primary fish pathogen which is the causative agent of bacterial hemorrhagic septicemia (motile aeromonad septicemia) (Austin and Austin, 1999; Chu and Lu, 2005). The disease is often associated with serious damage and economic losses in rainbow trout (Oncorhynchus mykiss) farming industry (Paniagua et al.,1990; Wang et al., 2003; Saglam et al., 2006). A. hydrophila produces several extracellular products such as proteases, haemolysins, aerolysin, cytolytic enterotoxins that are related with its pathogenicity (virulence) (Kingombe et al.,1999; 2010; Hu et al., 2012).Secreted extracellular hemolysin and cytolytic enterotoxin by bacteria are reported to be important for causing certain lytic activities in host cells (Watanabe et al.,2004; Uma et al., 2010). Additionaly, it has been shown that protein layers, O-antigens, fimbriae and outer membrane proteins of A. hydrophila play essential role of adherence of mechanism and contribute to colonization of fish tissue (Fang et al., 2004; Juarez et al., 2005; Khushiramani et al.,2007). The involvement of some virulence factors in Aeromonas spp. which were encoded by several genes, has been demostrated (Chacon et al., 2003; Xia et al., 2004). In the past, traditional microbiological and biochemical studies indicated that hemolytic and lytic activities were occured in virulent A. hydrophila strains (Kozaki et al., 1987; Santos et al., 1988). Moreover, despite the number of other studies performed on the house keeping genes to detect A. hydrophila such as 16 sRNA, entorotoxin (act), and DNA grase (gyrB), they were not able to confirm pathogenicity of bacteria (Ottaviani et al., 2011). Therefore, it is needed to developed more reliable, pathogen-specific and fast identification system to determine related virulence genes for identification of bacterial agent. With this method, not only fast identification can be done without much labor.For this reason, we aimed rapid identification of only pathogenic strains of A. hydrophila from clinical isolates in rainbow trout in comparison to the reference strain by showing presence of three positive PCR-amplicons of hemolysin (Hly+), cytolytic enterotoxin (AHCYTOEN+) and bacterial outer membrane protein (OmpTS+) gene determinants in the same sample.

Materials and Methods

Field Sample Collection

A total of 100 clinically observed infected fishes which ranged between 10-200 g from South-West Region of Turkey (Mugla-Fethiye) were collected for A. hydrophila between 2009 and 2011. Samples from skin ulcer, infected liver and kidney were taken by using sterile swabs. The ends of the swabs were cut off into both 15 mL falcon bottle containing 5 mL of transport medium (tryptic soy broth - TSB) and 5 mL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) and then transferred to the laboratory for DNA extraction.

Reference strain of A. hydrophila 19570 was obtained from American Type Culture Collection (ATCC), USA. Bacteria was grown in tryptic soy agar (TSA) plates and broths (TSB) at 22-25oC for 24 to 48 h. Stock cultures were maintained in a broth medium supplemented with glycerol at -20oC (Austin and Austin, 1999).

DNA Isolation

One mL of bacterial suspension from reference culture and swabs of field tissue were transferred to 2 mL centrifuge tubes, and centrifuged at 9000 x g for 5 min. Supernatants were discarded and the pellets were dissolved in 1 mL TE buffer (10 mM Tris-HCl, 1mM EDTA, pH 8.0). The genomic DNA was purified by using Qiagen DNA Extraction Kits (Qiagen, USA) as per the manufacturer's instructions.

Primers and PCR Amplification of Genes

Three pairs of primers (Table 1) were designed to detect targeted AHCYTOEN, OmpTS and Hly genes. The oligonucleotide primers were synthesized by Metabion (Germany).PCR amplification of AHCYTOEN gene was performed by modifying the method of Kingombe et al. (1999) by using DNA thermal cycle (Techne, Inc., USA). The reaction mixture contained of 50-100 ng of template DNA, 0.25 L of 0.5 U of Taq polymerase (Fermantas, USA), 5 L PCR buffer (50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, pH 8.3), 250 nmol/L of each dNTP, 5 mM of each primers. PCR were run under the following conditions: heat denatutation at 94oC for 2 min, primer annealing at56oC for 2 min and DNA extension at 72oC for 1 min in each cycle. PCR of OmpTS gene was amplified according to the protocols of Khushiramani et al. (2007) and Juarez et al. (2005). PCR amplification for Hly gene was performed using a total volume of 50 L of reaction mixture consistedof 5 L template DNA, 1 U of Taq polymerase (Fermantas),5 L buffer (50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl2, pH 8.3), 4 L of dNTPs (250 mol/L of each), 500 nmol/L of each primers. PCR were run under the following conditions; preheating at 95oC for 5 min followed by 30 cycles at 95oC for 2 min, 55oC 1 min and 72oC for 1 min, followed by 7 min final extension at 72oC.All PCR products were analysed by gel electrophoresis 1% agarose (Applichem, USA) containing0.6 g/mL ethidium bromide in TE buffer. DNA bands were visualized over a UV transilluminator at 254 nm (DNR geldoc, USA).


Total of 100 diseased fish samples were collected over the period of 2009 to 2011. In order to investigate whether those samples have been infected by pathogen A. hydrophila or not, direct PCR from their DNAs were amplified to show the targeted AHCYTOEN, OmpTS and Hly genes in less than 3 h. DNA extract from direct field tissue samples and reference stain were subjected to PCR amplifications using specific primers (Table 1), which were designed to detect pathogen A. hydrophila by PCR. All primer sequences were compared against each other and homology searches performed against the GeneBank database for sequence similarities using BLAST program (NCBI, USA).An amplimer of 232 bp for AHCYTOEN gene was obtained from both field and reference samples. Thypical positive amplicon results of AHCYTOEN gene in A. hydrophila are shown in Fig. 1. PCR products of 1008 bp of OmpTS gene were obtained from liver, kidney and skin samples and reference strains. The positive amplicons of OmpTS gene in A. hydrophila are shown in Fig. 2. An amplimer of 597 bp of Hly gene was obtained from both field and reference samples. Thypical positive amplicons of Hly gene to detect A. hydrophila are shown in Fig. 3. As a result, a total of 25 liver, 22 kidney and 21 skin lesion swabs samples were positive for all three virulence genes in A. hydrophila.


Use of virulence-associated genes as detection markers is very convenient and rapid method to identify organisms without requiring traditional microbiological culturing process. Previous studies have shown that production of hemolysin, aerolysin, cytolytic toxins and bacterial membrane receptors might individually contribute the virulence of A. hydrophila (Kingombe et al., 1999; Gonzales-Serrano et al., 2002; Ottaviani et al., 2011). However, it is suggested that proteolytic, haemolytic and cytotoxic activities of A. hydrophile could be changed upon in vitro passaging and temperature changes during culturing process (Morgan et al., 1985). In the last decade, hemolysin or outer bacterial membrane protein gene encoding studies indicated that hemolytic and/or adhesin activities occurred in the presence of genes in virulent A. hydrophila strains (Gonzales-Serrano et al., 2002; Khushiramani et al., 2007).In our study, six pairs of primers (Table 1) were generated and compared with other sequences in the GenBank database to display the ability of amplification efficiency. Our results confirmed that those primers were able to amplify three separate virulence genes in both reference strain and in direct liver, kidney and skin swabs in less than 3-4 h. We have shown that PCR amplification results of AHCYTOEN+, Hly+ and OmpTS+ genes from each separate swab could indicate only virulent A. hydrophila. These genes were unable to detect non- pathogenic A. hydrophila strains, which did not contain the cytotoxin and hemolysine genes. Therefore, we demonstrated that 68 of 100 samples are virulent-related genes as a result of existence of hemolysin, adherence and enterotoxin activities. Like Wang et al. (2003), our results supported the view that bacteria isolated from health fishes did not carry the hemolysin genes. Table 1: Primers used to detect pathogenic A. hydrophilaFurthermore, the studies of Khushiramani et al. (2007) and Juarez et al. (2005) have confirmed our OmpTS gene amplification findings that virulent A. hydrophila has adherence activity to attach host epithelial cells surface mediated by presence of membrane proteins. Moreover, these proteins are known as potentially important to vaccine components (Juarez et al., 2003). Fang et al. (2004) also showed the presence of major adhesion gene in virulent A. hydrophila. Lastly, Kingombe et al. (2010) showed the cytolitic enterotoxin gene in Aeromonas spp.In conclusion, we have developed rapid-multiplex gene markers method to detect only pathogenic A. hydrophila, which poses a threat to rainbow trout farms in Turkey. Further studies are needed to understand the pathogenesis of these bacteria and host relationship. The regional distribution of bacteria and analysis of nucleotide sequences in virulent isolates should also be studied to develop strain-specific DNA vaccine against this infection.

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Author:Cagatay, Ifakat Tulay; Sen, Evrim Beyhan
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:7TURK
Date:Apr 30, 2014
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